Generation and Characterization of CAR-T Cells.

CARs are synthetic receptors that link antigen binding to T-cell activation. Most CARs used in the clinic for treating cancer are second generation (2G) and comprise (i) a single chain variable fragment (scFv) that binds the target tumor antigen, (ii) a linker/hinge region, (iii) a transmembrane domain, (iv) a costimulatory endodomain, and (v) the endodomain of CD3 zeta. Our lab is focused on the development of function and safety-enhanced, next-generation CAR-T cells for the treatment of solid tumors. For example, we have designed switchable CARs that can be remotely turned on or off upon small molecule administration in order to mitigate toxicity or exhaustion. To address barriers to CAR-T cells in the solid tumor microenvironment, we are further developing rational coengineering strategies to support their function. While we have implemented non-viral tools like CRISPR/Cas9 knockout and knockin, adenine base editing, and transposon-based systems for T cell engineering in the lab, currently we mostly use lentivirus and retrovirus for our pre-clinical studies. Here, we present our most frequently used protocols, improved over many years in the lab, for the production and titration of lentivirus and retrovirus, as well as the purification, activation, transduction and expansion of both mouse and human CAR-T cells. In addition, we share protocols for our most commonly run in vitro assays for characterizing CAR-T cells, including for evaluating transduction efficiency, proliferation, phenotype, cytokine/chemokine production, cytotoxicity, and resistance to stress. Most of these protocols can also be applied to the production and characterization of T cell receptor (TCR)-engineered T cells. Finally, we explain how to set up and perform CAR-T cell transfer studies in subcutaneous tumor-bearing mice, both for syngeneic and xenograft models, and perform ex vivo analysis on tumor tissues post-treatment.